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121 → Gly) were inserted into the Bam HI site of pAcYM1-GST to produce GST-CAT and GST-CAT-KD, respectively. GST-CAT and GST-CAT-KD were produced in Sf9 cells with a baculovirus system [Y. Matsuura et al., J. Gen. Virol. 68, 1233 (1987)] and purified on a glutathione-Sepharose column (10). The cDNAs encoding the coiled-coil domain (amino acids 421 to 701), Rho-binding domain (amino acids 941 to 1075), and pleckstrin-homology domain (amino acids 1125 to 1388) were inserted into the Bam HI site of pGEX-2T to produce COIL, RB, and PH, respectively. COIL, RB, and PH were produced and purified from E. coli as described (10). The pEF-BOS-myc mammalian expression plasmids encoding CAT, CAT-KD, COIL, RB, and PH were constructed.
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A37. The labeled bands were visualized by an image analyzer (Fuji).
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-32P]ATP (14 to 540 mCi/mmol), 4 μg of myosin light chain, and 20 ng of Rho-kinase or 8 ng of GST-CAT with or without 1.5 μM GTP-γ-S·GST-RhoA (13). After incubation for 10 min at 30°C, the reaction mixtures were boiled in SDS-sample buffer and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). The labeled bands were visualized by an image analyzer (Fuji).
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3 cells onto 12-mm glass cover slips. After 4 days, the cells were deprived of serum for 24 hours in DMEM. Recombinant proteins were microinjected along with a marker protein (rabbit immunoglobulin G, 1 mg/ml) into the cytoplasm of cells. After microinjection, the cells were incubated at 37°C for 30 min. Actin and vinculin were visualized by tetramethylrhodamine B isothiocyanate (TRITC)-labeled phalloidin and an antibody to vinculin, respectively, as described (2). Nuclei were visualized by bisbenzimide.
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3 cells onto 12-mm glass cover slips and cultured for 1 day. Various plasmids were injected into the nucleus as described [A. Ridley et al., Cell 70, 401 (1992)]. After microinjection, the cells were incubated at 37°C for 3 hours. Actin was visualized with TRITC-labeled phalloidin as described (2).
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We thank M. Ito for chicken myosin light chain, S. Narumiya for C3 transferase, Y. Ono for the PKN cDNA, K. Umesono and R. Yu for critical reading of the manuscript, and M. Nishimura for secretarial assistance. Supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture of Japan and by grants from the Mitsubishi Foundation and Kirin Brewery Company.
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